The Regulatory Mechanism Of Endoplasmic Reticulum Stress In Cadmium-induced Autophagy Of Epithelioma Papulosum Cyprini Cell

Objective:Cadmium(Cd)is a toxic heavy metal,which can cause great harmness to the environment and human health.Recent studies have shown that cadmium exposure can induce autophagy.In the mammalian model,cadmium can induce autophagy in the ER stress pathway,whether endoplasmic reticulum stress is involved in the regulation of autophagy mechanism induced by cadmium stress is still unclear.Therefore,this study aims to investigate whether cadmium regulates autophagy in EPC cells through the unfolded protein reaction and calcium ion signaling pathway,so as to reveal the autophagy mechanism of EPC cells under cadmium stress and provide basic information for the treatment of fish diseases caused by heavy metals.Methods:1.The MTS method was used to detect the inhibitory effect of different concentrations of Cd Cl2 on the proliferation of EPC cells.2.We used flow cytometry and Hoechest 33342 staining to detect the effect of Cd Cl2 on EPC cell apoptosis.3.The main signaling molecules of autophagy and endoplasmic reticulum stress pathway Atg5,Atg7,p62,GRP78 and Ca MKK were cloned and the corresponding polyclonal antibodies were prepared.4.We used Western blot,Real-time quantitative PCR and gene silencing methods to detect the effect of Cd Cl2 on the expression of genes related to autophagy and endoplasmic reticulum stress in EPC cells.Results:We cloned autophagy and endoplasmic reticulum related genes of carp and prepared corresponding polyclonal antibodies based on the cloned c DNA sequences,RT-q PCR and Western blot results showed that cadmium stress significantly up-regulated the expression of autophagy(Atg5,Atg7,Beclin1,P62)and endoplasmic reticulum stress(GRP78,Ca MKK)related genes;Adding the endoplasmic reticulum stress inhibitor(4BPA)down-regulated the expression of cadmium-induced endoplasmic reticulum stress(GRP78,Ca MKK)and autophagy(Beclin1)related genes;Moreover,the autophagy level of EPC cells induced by cadmium decreased after GRP78 silencing.Conclusion:The unfolded protein reaction and Ca2+ signaling pathways in endoplasmic reticulum stress response are involved in the regulation of Cd-induced autophagy in EPC cells.